In both animals and plants, various types of programmed cell death (PCD) are required for the removal of cells that are dying, infected or no longer needed. Plant innate immunity relies on a specialized type of PCD called the hypersensitive response (HR-PCD) to restrict pathogens and prevent disease. Activation and execution of HR-PCD requires rapid cellular reprogramming and the coordination between different compartments within the cell. Interestingly, our results indicate that during HR-PCD, chloroplasts send out highly dynamic tubular extensions called "stromules" that physically connect chloroplasts to nuclei, possibly augmenting their communication. Very little is known about the cellular machinery required for stromule formation or the precise function of stromules during any biological process. The specific aims of the proposed research here will 1) employ candidate, genetic and proteomic approaches to identify novel factors required for stromule induction and will elucidate their biological role during HR-PCD and innate immunity. 2) The function of stromules during the generation and propagation of pro-cell death signals, such as hydrogen peroxide and salicylic acid, during HR-PCD will be revealed. 3) An in-depth study of chloroplast-to-nuclear associations using advanced microscopy techniques will shed light on the importance of inter-organellar communication during HR-PCD and innate immunity. The research outlined here will undoubtedly advance the field of innate immunity by providing researchers with a novel, cohesive model for HR-PCD that integrates the essential functions of chloroplasts, mitochondria and nuclei, and how they communicate to coordinate cellular reprogramming that leads to HR-PCD and innate immunity. Furthermore, these studies will reveal broader insights into the role of stromules in PCD and inter-organellar communication. Similar to how studies in multiple model systems, such as human cell lines, C. elegans, Drosophila and yeast, have all added to our understanding of PCD, the proposed studies here in plants will add new insight into conserved and divergent mechanisms that balance the life and death of a cell. PUBLIC HEALTH RELEVANCE: Inter-organellar communication is crucial for cells to coordinate the rapid cellular reprogramming that leads to the activation of programmed cell death (PCD). Plants use PCD as a defense mechanism to kill cells that are infected with potential pathogens. The study of PCD in model systems such as human cell lines, C. elegans, Drosophila and yeast, have contributed significantly towards our understanding of PCD in growth, development, defense and cancer. The proposed studies here in a plant model system will provide novel insights into conserved and divergent mechanisms that balance the life and death of a cell.